The human genome includes stretches of DNA composed of short tandem repeats (STRs). The analysis of such STRs is an important tool for genetic linkage studies, forensics, and new clinical diagnostics because STRs are abundant and their locations have been mapped in genomes.
A typical STR is less than 400 base pairs in length, and includes repetitive units that are two to seven base pairs in length. STRs can define alleles which are highly polymorphic due to large variations between individuals in the number of repeats. For example, four loci in the human genome CSF1PO, TPOX, THO1, and vWA (abbreviated CTTv) are characterized by an STR allele which differs in the number of repeats. Two repeating units are found at these loci: AATG for TPOX and THO1, and AGAT for CSF1PO and vWA.
In general, STR analysis involves generating an STR profile from a DNA sample, and comparing the generated STR profile with other STR profiles. Generating an STR profile typically involves dying or tagging STRs within a DNA sample, separating the tagged STRs within the sample using electrophoresis (applying an electric field), and recording the tagged STRs using a detector (e.g., a laser and a scanner).
One procedure for generating an STR profile uses an elongated gel plate (or slab gel) that is approximately 35 cm long. In general, this process (hereinafter referred to as “the gel plate process”) involves depositing a tagged DNA sample on an area of the gel plate, separating the STRs within the tagged DNA sample on the gel plate using electrophoresis, and scanning the gel plate with a detector to record the tagged STRs. Typically, the gel plate process requires two to three hours to complete.
Another procedure for generating an STR profile uses a capillary that is 50 to 75 microns in diameter. This process (hereinafter referred to as “the capillary process”) generally involves placing a tagged DNA sample at one end of a capillary, and drawing the sample through the capillary using electrophoresis to separate the STRs. A detector records the STRs by scanning a portion of the capillary.
Typically, STR separation is faster in the capillary process than in the gel plate process. In general, an increase in electrophoresis current results in an increase in STR separation speed, and a higher electrophoresis current typically can be applied to the capillary than to the gel plate because the capillary more easily dissipates heat (caused by the current) which would otherwise skew the separation results. A typical capillary process requires between 10 minutes and one hour to complete.
Another procedure for generating an STR profile uses a microchip (or chip) made of durable transparent glass or plastic. A typical microchip is a monolithic structure that is planar in shape. Such a microchip includes multiple pairs of channels (channel pairs) that run in a coplanar manner with the plane of the microchip. An individual STR separation can be performed at each channel pair. Each channel pair includes a long channel and a short channel. The short channel intersects the long channel near one end of the long channel and at a 90 degree angle. In some microchips, the short channel includes a jog where it intersects the long channel such that portions of the short channel are parallel but not co-linear. Typically, a microchip is formed using photolithography and chemical etching techniques to produce channel structures in fused silica wafers.
An STR separation process that uses a microchip (hereinafter referred to as “the microchip process”) generally involves orienting the microchip so that it and the channels within lie horizontally (i.e., perpendicular to the direction of gravity) and depositing a sample of tagged DNA over a hole in the upper surface of the microchip that connects with one end of the short channel of a channel pair. Next, the DNA sample is drawn horizontally through the short channel using electrophoresis such that STRs within the sample are partially separated along the short channel. Then, a portion of the sample at the intersection of the long and short channels is further separated along the long channel using electrophoresis. A detector records the STRs in a manner similar to that of the gel plate and capillary processes.
The microchip process provides advantages over the gel plate and capillary processes. First, the microchip process requires less time to complete than the gel plate and capillary processes because, in the microchip process, very large STRs (which impede STR separation in the gel plate and capillary processes) are removed from the DNA sample during electrophoresis along the short channel and thus do not impede STR separation along the long channel. Accordingly, STR separation along the long channel takes no more than a few minutes. Second, a microchip may include multiple channel pairs such that multiple samples can be separated and scanned simultaneously. Nevertheless, significant decreases in electrophoretic run-times would greatly increase the speed of STR analysis.
Conventional high-speed DNA genotyping using a microchip is described in an article entitled “High-Speed DNA Genotyping Using Microfabricated Capillary Array Electrophoresis Chips”, Analytical Chemistry, Vol. 69, No. 11, Jun. 1, 1997 on pages 2181 through 2186, the teachings of which are hereby incorporated by reference in their entirety. Ultra-high-speed DNA sequencing using capillary electrophoresis chips is described in an article entitled “Ultra-High-Speed DNA Sequencing Using Capillary Electrophoresis Chips”, Analytical Chemistry, Vol. 67, No. 20, Oct, 15, 1995 on pages 3676 through 3680, the teachings of which are hereby incorporated by reference in their entirety.